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Respirometry (calculation of metabolic
rate from changes in concentration of the gases -- oxygen and/or
carbon dioxide -- involved in cellular respiration) is simple in principle,
but can be a real headache to do correctly. John Lighton's book, Measuring Metabolic Rates - A Manual for Scientists is an excellent in-depth reference.
The major pitfalls stem
from the following issues:
- Organisms consume oxygen and emit CO2
-- but usually not in equal amounts. This creates volume changes -- and hence, fractional concentration changes --
that must be accounted for.
- Air-breathing organisms also emit water vapor, which increases total gas volume and dilutes the concentrations of O2 and CO2.
- Some organisms emit (either anteriorly or posteriorly) substantial
quantities of other gases, such as
methane, that affect concentrations of O2
and CO2.
- Volumes of respiratory gases are strongly affected by temperature and
pressure.
- The arrangement of the various elements of a respirometry system --
volume or flow meters, animal chambers, gas scrubbing units, and gas analyzers
-- determines the correct conversion equation.
- Rapid changes in gas exchange are buffered or 'hidden'
by the mixing characteristics of the respirometry system. To some extent, they also may be buffered within the organism: for example, CO2 emission can fluctuate with temperature changes, independent of metabolic events.
- Some gas exchange calculations require knowledge of the concentrations of two gas species (usually O2 and CO2). Unfortunately, these computations can be compromised because different kinds of gas analyzers respond at different rates. This is of particular concern if metabolism is changing rapidly.
- The energy equivalents of gas exchange rates differ between O2 and CO2, and
also with the metabolic fuel being consumed.
These issues -- and how LabAnalyst
deals with them -- are addressed in more detail below and on other pages in the Respirometry menu.
Note: if you are a
respirometry geek -- and who else would waste time reading this? -- you
may have noticed that I'm not using the correct abbreviations
for rates of oxygen consumption and carbon dioxide production. Proper
usage includes a dot over the V in VO2 and VCO2 to
indicate rates instead of volumes, e.g.:
Alas, I haven't found a convenient way
to make the 'Vee-dot' symbol in html. |
Open versus constant
volume systems:
There are two methods in routine use for
measuring rates of gas exchange: open system respirometry and constant
volume ('closed system') respirometry. In the former, a continuous flow of air (or
other fluid) moves past the animal, and you measure the incurrent vs. excurrent difference in gas
concentrations. Most of the calculations
and discussion on this page concern open systems; this page contains more information on open-system design.
In constant volume respirometry (CVR), the organism
is placed in a sealed chamber, and over time its respiration changes the
gas concentrations in the chamber. You measure rates of gas exchange
by determining gas concentrations (O2 and/or
CO2) at the start and end of a period of
measurement, and then using the volume, the cumulative (initial versus final) difference in concentrations,
and the elapsed time to compute the average rate of change. The most
straightforward way to handle constant volume calculations with LabHelper and LabAnalyst
is as follows:
- First, collect samples of 'initial' and 'final' gas from the animal
chamber(s) and inject them through a gas analyzer while continually recording
the concentration (in %) with LabHelper or some other system (e.g., Sable ExpeData).
Between injections, flush the analyzer with reference gas (or fluid).
You should get a data file with a series of 'peaks', one for each injection
of 'final' gas (or fluid). See this diagram for a convenient way to set up a system for rapid analysis of multiple CVR samples.
- Next, in LabAnalyst, use the
baseline function to set the 'initial' values at zero. The 'final'
values now show the % change during the measurement period.
- Finally, for each peak, find the maximum deflection from baseline with
the 'maximum value '
option in the ANALYZE menu.
Click the 'C.V.R. options...'
button to set up the constant volume calculations.
A more versatile method for computing C.V.R. is in the SPECIAL menu. This offers more options than the C.V.R. window in the maximum value calculator
Note that this approach can only provide an average rate of gas exchange: it does not reveal any temporal changes in metabolism during the measurement period. However, if you have an oxygen or CO2 sensor within the sealed chamber (or if the respiratory fluid is circulated past a sensor in a closed circuit), you can measure the decline in pO2 or increase in pCO2 continuously, and then take the derivative of the change to get time-specific metabolic rates.
With either open system or constant volume respirometry,
if your animal is an air-breather you need to think about the possibility
that its overall metabolism involves gases other than water, O2, and CO2. For example, ruminants and other herbivores may emit large quantities of methane and other organic gases as a consequence of their digestive physiology.
These result largely from the microbial fermentation in
specialized gut regions. However, the effect is
not limited to plant-eating species with specialized fermentation chambers. Any animal with bacterial flora in the gut will likely produce organic gases (humans are well known for this...).
Predators such as snakes can emit substantial amounts of various
decomposition gases (H2, etc.) during the digestive breakdown of prey.
These
additional gases have two possible effects, both of which can
compromise measurement accuracy:
- With any gas analyzer, 'extra' gases dilute the
concentrations of O2 and CO2. Unless you quantify this, you might not calculate
VO2 or CO2
accurately. Most animals don't emit enough of these gases to cause much of a dilution problem, but you need to be aware of the potential.
- With some oxygen analyzers that have high-temperature measurement cells
(like the zirconia cells used by Applied Electrochemistry/Ametek S-3As),
another error can result when organic gases oxidize -- combust
-- in the cell. The S-3A's cells operate at more than 600 °C, so
there is plenty of potential for this to occur. Oxidization removes
oxygen from the gas stream. It also produces CO2,
water, and other reaction products that lower the concentration of the
remaining O2 and further increase the error (generating an artifactually high VO2). And any liquid water entering the cell, e.g., from condensation, can cause explosive vaporization and thermal shock that breaks the sensor.
If your animal does produce substantial quantities of non-standard respiratory gases, the solution is to remove the problematic gas species
from analysis air by using an appropriate scrubbing filter upstream of the
gas analyzer(s). This, too, incurs operational penalties, such as reduced response time and more scrubbing tubes to keep fresh.
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